The fate map of the chick forelimb-bud and its bearing on hypothesized developmental control mechanisms

Summary Carbon particles and isotopic quail grafts were used as markers to study the salient features of the fate map of the chick forelimb between stages 20 and 27. The grafting technique confirmed the reliability of the carbon method: they both revealed striking asymmetries in which apical mesodermal tissue was progressively displaced in a proximal direction (as would be expected on the basis of growth by net apical addition of tissue) but also in a preaxial direction, while postaxial tissue became elongated in the direction of limb outgrowth. Ectoderm showed a similar preaxial-postaxial asymmetry but became displaced from initially underlying mesoderm. In marked contrast to mesoderm, distal ectoderm remained at a constant distance from the apical ectodermal ridge (or became incorporated into it), thus implying that the ectodermal sheet is anchored distally and grows by uniform stretching proximally. Within the ectoderm itself, the outer peridermal layer is displaced distally relative to the underlying epidermal basal layer. Peripheral mesoderm showed patterns of displacement which were intermediate between those of ectoderm and chondrogenic core mesoderm. It is argued that such morphogenetic phenomena are integral components of developmental mechanisms of significance in the control of pattern generation. Implications of the interpretation and use of the fate map in relation to theories of limb development, particularly those based on mechanisms defined in terms of limb axes, are reviewed.     

A role for epithelial-mesenchymal interactions in tail growth/morphogenesis and chondrogenesis in embryonic mice

Neurulation involves development from primary germ layers before any differentiation of embryonic mesenchyme. Subsequently, secondary organogenesis is via epithelial-mesenchymal interaction. It is unclear whether formation of the caudal body axis and tail bud in vertebrate embryos is by temporal and causal extension of primary neurulation, by secondary neurulation, or by secondary induction (epithelial-mesenchymal interactions) as seen in organogenesis of the limb buds, kidneys, heart and other embryonic regions. Reports of a ventral ectodermal ridge (VER) associated with tail bud development in rodent embryos imply that tail bud development may share features with limb bud development, in which the apical ectodermal ridge (AER) directs limb bud outgrowth and skeletal patterning. Organ culture or grafting to the chorioallantoic membranes of host chick embryos, of tail bud mesenchyme with or without tail epithelium, demonstrates that both survival and growth of tail mesenchyme depend on the presence of tail epithelium. Initiation of chondrogenesis of tail mesenchyme was similarly dependent on tail epithelium until 10.5 days of gestation, which is when the VER is at its maximal extent. Initiation of myogenesis was independent of the presence of tail epithelium. These results are discussed in relation to the similarity of tail bud to limb bud developed, and to the different mechanisms employed in differentiation of the cranial and caudal ends of vertebrate embryos. Secondary induction of the caudal body region is argued to be fundamental in vertebrate embryogenesis.     

Loss of the Tg737 protein results in skeletal patterning defects.

Tg737 mutant mice exhibit pathologic conditions in numerous tissues along with skeletal patterning defects. Herein, we characterize the skeletal pathologic conditions and confirm a role for Tg737 in skeletal patterning through transgenic rescue. Analyses were conducted in both the hypomorphic Tg737(orpk) allele that results in duplication of digit one and in the null Tg737(delta2-3betaGal) allele that is an embryonic lethal mutation exhibiting eight digits per limb. In early limb buds, Tg737 expression is detected throughout the mesenchyme becoming concentrated in precartilage condensations at later stages. In situ analyses indicate that the Tg737(orpk) mutant limb defects are not associated with changes in expression of Shh, Ihh, HoxD11-13, Patched, BMPs, or Glis. Likewise, in Tg737(delta2-3betaGal) mutant embryos, there was no change in Shh expression. However, in both alleles, Fgf4 was ectopically expressed on the anterior apical ectodermal ridge. Collectively, the data argue for a dosage effect of Tg737 on the limb phenotypes and that the polydactyly is independent of Shh misexpression.     

Ventral ectodermal ridge and ventral ectodermal groove: two distinct morphological features in the developing rat embryo tail

The ventral ectodermal ridge (VER) is a thickening of the surface ectoderm on the ventral side of the embryonic tail which resembles the apical ectodermal ridge of the limb bud. The morphological characteristics of the ventral part of the embryo tail were investigated in 10.5- to 14-day rat embryos by light microscopy of serial semithin sections and by scanning and transmission electron microscopy. In 10.5- to 11.5-day embryos the thickening of the ventral surface ectoderm includes the complete ventral midline of the tail and can be divided into two parts. The posterior part is elevated and represents the ventral ectodermal ridge. The anterior part is, in contrast to the ridge, concave, and we have termed it the ventral ectodermal groove (VEG). The cloacal membrane is located at its anterior end. Contacts between the VER and the mesenchymal cells are visible until an intact basal lamina is formed at 11.5 days. Similarly, the VEG is connected by elongated cell processes with the ventral part of the tail gut. Gap junctions are present between the apical parts of ridge and groove cells. The VEG flattens and disappears in 12-day embryos. At this stage the ridge is at its maximum height, simultaneously undergoing extensive cell death. The VER is no longer visible in 14-day rat embryos.     

The distribution of mesenchyme proteoglycan (PG-M) during wing bud outgrowth

Summary This study utilizes immunofluorescence to describe the distribution of several extracellular matrix molecules in the chick embryo during the process of limb outgrowth and the formation of precartilage condensations. A large chondroitin sulfate proteoglycan (PG-M) is detected at the wing level at Hamburger and Hamilton stage 14 in and under the dorsal ectoderm, and is associated with the basement membranes around the neural tube, notochord and pronephros, but not with other basement membranes. The galactose-specific leetin, peanut agglutinin (PNA), has a similar distribution except that it also binds to the dorsal side of the neural tube. PG-M is not detected in the limb mesenchyme until after stage 17, when it is present in the distal region, as is PNA-binding material. With further development of the wing bud, PG-M is present in the subectodermal mesenchyme, the mesenchyme at the distal tip and in the prechondrogenic core. After stage 22 PNA-binding material becomes localized in the prechondrogenic core, the basement membranes under the apical ectodermal ridge, and the ventral sulcus. The distribution of these components (PG-M and PNA binding material) overlaps, but differs from that of type I collagen and fibronectin and basement membrane components, such as laminin, basement membrane heparan sulfate proteoglycan, and type IV collagen. Tenascin, on the other hand, is not detected in the limb bud until stage 25, after the appearance of cartilage matrix components such as type II collagen and cartilage proteoglycan (PG-H). These results are considered in relation to the formation of precartilage aggregates, and indicate that PNA binds to components in precartilage aggregates other than PG-M or tenascin.     

The apical ectodermal ridge, fibroblast growth factors (FGF-2 and FGF-4) and insulin-like growth factor I (IGF-I) control the migration of epidermal melanoblasts in chicken wing buds

The role of the apical ectodermal ridge and of fibroblast growth factors FGF-2 and FGF-4 and of the insulin-like growth factor I (IGF-I) in the control of the migration of epidermal melanoblasts was investigated using quail-chicken chimeras. Wing buds of a strain of unpigmented chicken were microsurgically modified in several ways (ablation, displacement or implantation of additional apical ectodermal ridges, implantation of grafts devoid of apical ectodermal ridges, ectopic application of growth factors) and received grafts containing quail neural crest cells. The distribution of the epidermal melanoblasts which had differentiated from the quail grafts revealed that both the apical ectodermal ridge and the growth factors invariably caused the migration of epidermal melanoblasts towards them. This leads to the conclusion that the presence of the apical ectodermal ridge is the sufficient condition to direct the migration of epidermal melanoblasts within the avian embryonic wing bud. Furthermore, FGF-2 and IGF-I and to a lesser extent FGF-4 play a decisive role in directing the migration of epidermal melanoblasts within chicken wing buds and are likely to be involved in the molecular cascade by means of which the apical ectodermal ridge controls the migration of epidermal melanoblasts.     

Btg1 and Btg2 gene expression during early chick development

Btg/Tob genes encode for a new family of proteins with antiproliferative functions, which are also able to stimulate cell differentiation. Btg1 and Btg2 are the most closely related members in terms of gene sequence. We analyzed their expression patterns in avian embryos by in situ hybridization, from embryonic day 1 to 3. Btg1 was distinctively expressed in the Hensen's node, the notochord, the cardiogenic mesoderm, the lens vesicle, and in the apical ectodermal ridge and mesenchyme of the limb buds. On the other hand, Btg2 expression domains included the neural plate border, presomitic mesoderm, trigeminal placode, and mesonephros. Both genes were commonly expressed in the myotome, epibranchial placodes, and dorsal neural tube. The results suggest that Btg1 and Btg2 are involved in multiple developmental processes. Overlapping expression of Btg1 and Btg2 may imply redundant functions, but unique expression patterns suggest also differential regulation and function.     

Vertebrate limb development: from Harrison's limb disk transplantations to targeted disruption of Hox genes

Various animal organs have long been used to investigate the cellular and molecular nature of embryonic growth and morphogenesis. Among those organs, the tetrapod limb has been preferentially used as a model system for elucidating general patterning mechanisms. At the appropriate time during the embryonic period, the limb territories are first determined at the right positions along the cephalocaudal axis of the animal body, and soon the limb buds grow out from the flanks as mesenchymal cell masses covered by simple ectoderm. The position, number, and identity of the limbs depend on the expression of specific Hox genes. Limb morphogenesis occurs along three axes, which become gradually fixed: first the anteroposterior axis, then the dorsoventral, and finally the proximodistal axis, along which the bulk of limb growth occurs. Growth of the limb in amniotes depends on the formation of the apical ectodermal ridge, which, by secreting many members of the fibroblast growth factors family, attracts lateral plate and somitic mesodermal cells, keeps these cells in the progress zone proliferating, and prevents their differentiation until an appropriate time period. Mutual interactions between mesoderm and ectoderm are important in the growth process, and signaling regions have been identified, such as the zone of polarizing activity, the dorsal limb ectoderm, and the apical ectodermal ridge. Several molecules have been found to play leading roles in various biological processes relevant to morphogenesis. Besides its intrinsic merit as a model for unraveling the mechanisms of development, the limb deserves considerable clinical interest because defects of limb development are the most common single category of congenital abnormalities.     

Vertebrate limb development: from Harrison’s limb disk transplantations to targeted disruption of<Emphasis Type="Italic"> Hox</Emphasis> genes

Various animal organs have long been used to investigate the cellular and molecular nature of embryonic growth and morphogenesis. Among those organs, the tetrapod limb has been preferentially used as a model system for elucidating general patterning mechanisms. At the appropriate time during the embryonic period, the limb territories are first determined at the right positions along the cephalocaudal axis of the animal body, and soon the limb buds grow out from the flanks as mesenchymal cell masses covered by simple ectoderm. The position, number, and identity of the limbs depend on the expression of specific Hox genes. Limb morphogenesis occurs along three axes, which become gradually fixed: first the anteroposterior axis, then the dorsoventral, and finally the proximodistal axis, along which the bulk of limb growth occurs. Growth of the limb in amniotes depends on the formation of the apical ectodermal ridge, which, by secreting many members of the fibroblast growth factors family, attracts lateral plate and somitic mesodermal cells, keeps these cells in the progress zone proliferating, and prevents their differentiation until an appropriate time period. Mutual interactions between mesoderm and ectoderm are important in the growth process, and signaling regions have been identified, such as the zone of polarizing activity, the dorsal limb ectoderm, and the apical ectodermal ridge. Several molecules have been found to play leading roles in various biological processes relevant to morphogenesis. Besides its intrinsic merit as a model for unraveling the mechanisms of development, the limb deserves considerable clinical interest because defects of limb development are the most common single category of congenital abnormalities.     

Insulin improves survival but does not maintain function of cultured chick wing bud apical ectodermal ridge.

Previously we demonstrated that high levels of insulin (5 micrograms/ml) permit the survival of isolated chick apical ectodermal ridge in culture (Boutin and Fallon, Dev. Biol., 104:111-116, 1984). Here we address whether lower levels of insulin or insulin-like growth factors (IGFs) can also improve the survival of cultured apical ectodermal ridge and whether ridge function is maintained along with ridge survival. Neither IGF I nor IGF II (100 ng/ml) decreased ridge cell death; however, cell death was significantly decreased with 50 ng/ml insulin. No further improvement was obtained in the presence of both IGF I and insulin. These data suggest that insulin improved the survival of the isolated apical ectodermal ridge by binding its own receptor. To test for the maintenance of function, stage 20 ridges were cultured for 0, 6, 12, 18, or 24 hr with or without insulin (5 micrograms/ml or 5 ng/ml) and used to make recombinant limbs. Isolated ridges cultured for 12 hr or more produced fewer outgrowths and these were rarely distally complete. The medium in which the ridges had been cultured did not influence ridge activity, despite the major differences in cell survival. Recombinants made with ridges cultured with limb mesoderm for 18 hr did not yield outgrowths as often as those with freshly isolated ridges, but most of the limbs that did form were distally complete. These results suggest that the decline in function of cultured, isolated apical ectodermal ridge was not due merely to ridge cell death but rather, at least in part, to its separation from limb mesoderm.     

Insulin improves survival but does not maintain function of cultured chick wing bud apical ectodermal ridge

Previously we demonstrated that high levels of insulin (5 μg/ml) permit the survival of isolated chick apical ectodermal ridge in culture (Boutin and Fallon, Dev. Biol., 104:111–116, 1984). Here we address whether lower levels of insulin or insulin-like growth factors (IGFs) can also improve the survival of cultured apical ectodermal ridge and whether ridge function is maintained along with ridge survival. Neither IGF I nor IGF II (100 ng/ml) decreased ridge cell death; however, cell death was significantly decreased with 50 ng/ml insulin. No further improvement was obtained in the presence of both IGF I and insulin. These data suggest that insulin improved the survival of the isolated apical ectodermal ridge by binding its own receptor. To test for the maintenance of function, stage 20 ridges were cultured for 0, 6, 12, 18, or 24 hr with or without insulin (5 μg/ml or 5 ng/ml) and used to make recombinant limbs. Isolated ridges cultured for 12 hr or more produced fewer outgrowths and these were rarely distally complete. The medium in which the ridges had been cultured did not influence ridge activity, despite the major differences in cell survival. Recombinants made with ridges cultured with limb mesoderm for 18 hr did not yield outgrowths as often as those with freshly isolated ridges, but most of the limbs that did form were distally complete. These results suggest that the decline in function of cultured, isolated apical ectodermal ridge was not due merely to ridge cell death but rather, at least in part, to its separation from limb mesoderm. © 1992 Wiley-Liss, Inc.     

A splice variant of CD44 expressed in the apical ectodermal ridge presents fibroblast growth factors to limb mesenchyme and is required for limb outgrowth

Signals from the apical ectodermal ridge (AER) of the developing vertebrate limb, including fibroblast growth factor-8 (FGF-8), can maintain limb mesenchymal cells in a proliferative state. We report here that a specific CD44 splice variant is crucial for the proliferation of these mesenchymal cells. Epitopes carried by this variant colocalize temporally and spatially with FGF-8 in the AER throughout early limb development. A splice variant containing the same sequences expressed on model cells binds both FGF-4 and FGF-8 and stimulates mesenchymal cells in vitro. When applied to the AER, an antibody against a specific CD44 epitope blocks FGF presentation and inhibits limb outgrowth. Therefore, CD44 is necessary for limb development and functions in a novel growth factor presentation mechanism likely relevant in other physiological and pathological situations where a cell surface protein presents a signaling molecule to a neighboring cell.     

Retinoic acid specifically downregulates Fgf4 and inhibits posterior cell proliferation in the developing mouse autopod

Retinoic acid, when administered to pregnant mice on d 11·0 of gestation, causes limb skeletal abnormalities consisting of reduced digital number, shortening of the long bones and delayed ossification. We show here that these effects are correlated with a decrease in cell proliferation within 5 h of retinoic acid administration, specifically in the posterior half of the distal limb bud mesenchyme, from which the distal skeletal elements are generated. There is a specific downregulation of Fgf4 , a gene known to be involved in limb bud outgrowth and expressed only in the posterior part of the apical ectodermal ridge; Fgf8 , which is expressed throughout the apical ectodermal ridge, is unaffected. The reduction in Fgf4 expression is not accompanied by downregulation of Shh , nor of its receptor and downstream target gene Ptc , suggesting that the skeletal reduction defects induced by retinoic acid are mediated specifically by FGF4-induced skeletogenic mesenchymal cell proliferation.     

Lectins selectively label cartilage condensations and the otic neuroepithelium within the embryonic chicken head

Cartilage morphogenesis during endochondral ossification follows a progression of conserved developmental events. Cells are specified towards a prechondrogenic fate and subsequently undergo condensation followed by overt differentiation. Currently available molecular markers of prechondrogenic and condensing mesenchyme rely on common regulators of the chondrogenic program that are not specific to the tissue type or location. Therefore tissue‐specific condensations cannot be distinguished based on known molecular markers. Here, using the chick embryo model, we utilized lectin labeling on serial sections, demonstrating that differential labeling by peanut agglutinin (PNA) and Sambucus nigra agglutinin (SNA) successfully separates adjacently located condensations in the proximal second pharyngeal arch. PNA selectively labels chick middle ear columella and basal plate condensation, whereas SNA specifically marks extracolumella and the ventro‐lateral part of the otic capsule. We further extended our study to examine lectin‐binding properties of the different parts of the inner ear epithelium, neural tube and notochord. Our results show that SNA labels the auditory and vestibular hair cells of the inner ear, whereas PNA specifically recognizes the statoacoustic ganglion. PNA is also highly specific for the floor plate of the neural tube. Additionally, wheat germ agglutinin (WGA) labels the basement membrane of the notochord and is a marker of the apical‐basal polarity of the cochlear duct. Overall, this study indicates that selective lectin labeling is a promising approach to differentiate between contiguously located mesenchymal condensations and subregions of epithelia globally during development.     

An electron microscopic study of periderm cell development in mouse limb buds

Summary Development of periderm cells covering fore-and hindlimb buds of mouse em`ryos was observed by scanning and transmission electron microscopy at half day intervals from day 9.5 to 12.5 of gestation (vaginal plug=day 0).At day 9.5, the epidermis is single layered. Occasional periderm cells are present at day 10.5. By day 11.5 a complete layer of periderm cells has covered the entire limb bud.By scanning electron microscopic observation, periderm cells covering the apical ectodermal ridge (AER) are characterized by a small surface size and an elongated polygonal shape with the long axis parallel to the antero-posterior contour of the apical rim. Periderm cells covering the dorsal and ventral surfaces of the limb bud are relatively large and have a polygonal surface shape.The periderm covering the apical tip reflects well the developmental state of the AER. Hence, it is possible to estimate the development of the AER by observing the surface features of the apical periderm by scanning electron microscopy.This work was supported by Scientific research Grant No. 348082 from the Ministry of Education, Japan     

On the migration of epidermal melanoblasts in the avian embryonic wing bud

Summary After heterotopic grafting of quail neural crest cells to the wing buds of embryos of an unpigmented chicken strain, epidermal melanocytes of donor origin are found almost exclusively distal from the graft in the host's epidermis. This directed cell migration ceases, if the apical ectodermal ridge (together with a small amount of subridge mesoderm) is removed from the operated wing buds or if impermeable materials are interposed between it and the rest of the wing bud. Under these conditions epidermal melanocytes are found not only distal from but also proximal to the grafts. From this it may be deduced that the apical ectodermal ridge directs the migration of epidermal melanoblasts in the avian embryonic wing bud, possibly by a chemotactic mechanism. The presence or absence of the apical ectodermal ridge had no observable effect on the migratory behaviour of other neural crest derived cell populations (Schwann cells and non-epidermal melanocytes) in the wing bud. This shows that the apical ectodermal ridge specifically influences epidermal melanocytes.This work was supported by the Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung (P 4680)     

Ultrastructural localization of cholinesterase uring chondrogenesis and myogenesis in the chick limb bud

Summary Cholinesterase (ChE) is transiently expressed in undifferentiated embryonic cells. In the chick limb bud ChE-activity was found in the apical ectodermal ridge and in the subridge mesenchyme. The reaction was localized in the perinuclear cisterna, in an extensive network of narrow profiles of endoplasmic reticulum (ER), and in the Golgi complexThe chondroblasts emerging from the subridge mesenenyme, also showed strong ChE-activity. During differentiation the enzyme first disappeared from the Golgi zone. Then, the narrow ChE-positive ER was successively replaced by ChE-negative extended rough ER characteristic for the differentiated chondrocyte.The myoblasts showed weak ChE-activity with the same ultrastructural localization as in other mesenchymal cells. After fusion the myotubes exhibited strong ChE-activity in the perinuclear cisterna and the developing sarcoplasmic reticulum. In later stages of myogenesis the myoblasts were closely attached to the myotubes and had lost their ChE-activity.During mitosis of ChE-positive cells, ChE-activity was retained in fragments of perinuclear cisterna and ER. In ChE-active mesenchymal cells and chondroblasts we observed specialized contact zones between ER and plasma membrane. ChE-active cisternae of ER run parallel to the plasma membrane with a gap of approximately 10–15 nm. We discuss a possible function of a cholinergic system during morphogenesis.